INTERNATIONAL JOURNAL OF ONCOLOGY 44: 1529-1538, 2014 Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cells ELIZABETH E. SWEENEY, PING FAN and V. CRAIG JORDAN Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA Received December 12, 2013; Accepted December 30, 2013 DOI: 10.3892/ijo.2014.2329 Abstract. Models of long-term estrogen-deprived breast receptor (ER)-positive cell line MCF-7 that is highly sensitive cancer cells are utilized in the laboratory to mimic clinical to E2-stimulated growth (1). The MCF-7:5C and MCF-7:2A aromatase inhibitor-resistant breast cancer and serve as a subclones are derived from the parental MCF-7 cell line tool to discover new therapeutic strategies. The MCF-7:5C through long-term E2 deprivation (1-4). MCF-7:5C cells and MCF-7:2A subclones were generated through long-term express wild-type ER at a higher level than the parental line, estrogen deprivation of estrogen receptor (ER)-positive MCF-7 and are progesterone receptor (PR)-negative (3). These cells cells, and represent anti-hormone-resistant breast cancer. grow in the absence of E2, and do not respond to 4-hydroxy- MCF-7:5C cells paradoxically undergo estrogen-induced tamoxifen (4-OHT) (2,3). MCF-7:2A cells can induce apoptosis within seven days of estrogen (estradiol, E2) treat- expression of PR and express both wild-type (66 kDa) and ment; MCF-7:2A cells also experience E2-induced apoptosis mutant (77 kDa) ER (4,5). The mutant ER contains a repeat but evade dramatic cell death until approximately 14 days of of exons 6 and 7 and cannot bind E2 nor anti-estrogens; it is treatment. To discover and define the mechanisms by which expressed 4- to 10-fold lower than the wild-type ER (6). The MCF-7:2A cells survive two weeks of E2 treatment, systematic total ER level of MCF-7:2A cells is higher than in parental experiments were performed in this study. The data suggest MCF-7 cells, and they also grow in E2-free media. 4-OHT and that MCF-7:2A cells employ stronger antioxidant defense pure anti-E2 are able to block their growth (4,5). mechanisms than do MCF-7:5C cells, and that oxidative stress In addition to the different responses to anti-E2 observed is ultimately required for MCF-7:2A cells to die in response in MCF-7:5C versus MCF-7:2A cells, they also have different to E2 treatment. Tumor necrosis factor (TNF) family member apoptotic responses to E2. The MCF-7:5C cells undergo apop- activation is also essential for E2-induced apoptosis to occur tosis and die during the first week of E2 treatment, whereas the in MCF-7:2A cells; upregulation of TNFα occurs simultane- MCF-7:2A cells die later, after two weeks of E2 treatment (7). ously with oxidative stress activation. Although the unfolded MCF-7:5C cell response to estrogens and anti-estrogens has protein response (UPR) signaling pattern is similar to that been extensively studied in our lab; the data show that these in MCF-7:5C cells, it is not sufficient to cause cell death in cells undergo E2-induced apoptosis through mechanisms asso- MCF-7:2A cells. Additionally, increased insulin-like growth ciated with endoplasmic reticulum stress (ERS) and oxidative factor receptor β (IGF-1Rβ) confers a mechanism of growth stress (8,9). Thus far, there has been less focus on the clas- and anti-apoptotic advantage in MCF-7:2A cells. sification and mechanisms of the MCF-7:2A response. Network enrichment analyses done using gene arrays Introduction in timecourse experiments show overexpression of apop- totic- and stress-related pathways in the MCF-7:5C cells Aromatase inhibitor-resistant breast cancer cells are modeled after 24-96 h of E2 treatment; however, these analyses show in vitro by long-term E2-deprived breast cancer cell lines. the MCF-7:2A cells expressing more genes associated with The MCF-7:WS8 cell line represents a clone of the estrogen glutathione metabolism during this time period of E2 exposure (Fig. 1). This suggests that the two cell lines respond to E2 treatment using different signaling pathways. The MCF-7:5C cells respond by quickly inducing apoptosis, while the anti- Correspondence to: Professor V. Craig Jordan, Lombardi oxidant pathway may be more relevant to the MCF-7:2A cells. Comprehensive Cancer Center, Vincent T. Lombardi Chair of Experiments were designed to interrogate the apoptotic, stress Translational Cancer Research, Georgetown University Medical and antioxidant pathways in both cell lines to distinguish Center, 3970 Reservoir Rd NW, Research Building, Suite E501, signaling mechanisms in response to E2. Washington, DC 20057, USA The concept of E2-induced death is important because E-mail: [email protected] of its clinical relevance. A clinical study published in 2009 (10) compared two doses of E2 for second-line treatment after Key words: oxidative stress, estrogen deprivation, breast cancer, breast cancer patients had failed aromatase inhibitor therapy. insulin-like growth factor receptor, glutathione The authors showed that after long-term anti-hormone therapy, no response is lost with the lower dose of E2; overall 1530 SWEENEY et al: E2-INDUCED APOPTOSIS IN ESTROGEN-DEPRIVED BREAST CANCER Figure 1. Network enrichment analysis for MCF-7:WS8, MCF-7:5C and MCF-7:2A cells. Global gene arrays were performed to compare activated gene networks associated with 1 nM E2 treatment in the cell lines. Genes were analyzed after 2-24 and 24-96 h treatment. about 30% of women responded to E2 treatment. The goal of 100 µM), or E2 (1 nM) + BSO (100 µM). DNA content was this study is to uncover the mechanisms preventing the other measured as previously described (11). 70% of patients from responding, and perhaps find ways to circumvent their resistance. To this end, MCF-7:2A cells were Western blot analysis. Total MAPK (#9102), phosphorylated used as a model for E2-deprived breast tumors with the ability MAPK (#9101), total AKT (#9272), phosphorylated AKT to evade E2-induced apoptosis in the clinic. (#4051L), total eIF2α (#9722S), phosphorylated eIF2α (#9721S), and IRE1α (#3294S) antibodies were all purchased Materials and methods from Cell Signaling Technology (Beverly, MA, USA). IGF-1Rβ antibody (sc-713) was purchased from Santa Cruz Cell culture. All cell lines were cultured in phenol red-free Biotechnology (Santa Cruz, CA, USA). β-actin loading control RPMI-1640 media supplemented with 10% charcoal-stripped antibody (A5441) was purchased from Sigma-Aldrich. fetal bovine serum (SFS). Media and treatments were replaced Proteins were harvested from cells using cell lysis buffer every three days. Estradiol (E2) (Sigma-Aldrich, St. Louis, (Cell Signaling Technology) supplemented with Protease MO, USA), buthionine sulfoximine (BSO) (Sigma-Aldrich), Inhibitor Cocktail Set I and Phosphatase Inhibitor Cocktail and combinations were dissolved in ethanol and then in media. Set II (Calbiochem). Bicinchoninic acid (BCA) assay was AG1024 (Calbiochem, San Diego, CA, USA) was dissolved in used to quantify total protein content (Rio-Rad Laboratories, DMSO and then in media. Hercules, CA, USA). Protein (50 µg) was probed and visual- ized as previously described (11). DNA assays. MCF-7:WS8, MCF-7:5C and MCF-7:2A cells were harvested after 7 or 14 days treatment with vehicle Cell cycle analysis. MCF-7:2A cells were cultured in dishes -9 -4 -9 (0.1% ethanol), E2 (10 mol/l, 1 nM), BSO (10 mol/l, and treated with vehicle (0.1% ethanol) or E2 (10 mol/l, INTERNATIONAL JOURNAL OF ONCOLOGY 44: 1529-1538, 2014 1531 1 nM). Cells were harvested after 24 h, fixed in 75% ethanol on and IRE1α by 72 h of E2 treatment, indicating activated UPR. ice, stained with propidium iodide and sorted using FACS flow Though MCF-7:2A cells show a slightly higher basal p-eIF2α cytometry (Becton Dickinson, San Jose, CA, USA). Results level, no differences in UPR activation can be seen between were analyzed using CellQuest software. the two cell lines. RT-PCR. Cells were harvested using TRIzol, and RNA MCF-7:5C and MCF-7:2A estrogen-induced apoptosis. To deter- was isolated using RNeasy mini kit (Qiagen, Valencia, CA, mine whether MCF-7:2A cells experience apoptosis through USA). RNA was reverse transcribed to cDNA using a kit the same mechanism as MCF-7:5C cells, RT-PCR was used to (Applied Biosystems, Foster City, CA). SYBR-Green (Applied quantify mRNA levels of apoptosis-related genes. MCF-7:5C Biosystems) was used for quantitative real-time polymerase cells noticeably upregulate LTA (4.19±1.92 fold change), LTB chain reaction (RT-PCR) in a 7900HT Fast Real-Time PCR (5.39±1.82), TNFα (9.40±3.86), and BCL2L11 (6.06±0.87) after system (Applied Biosystems). 72 h of E2 treatment, while MCF-7:2A cells show no major changes during this time period (Fig. 4A). MCF-7:2A cells Glutathione assay. Cells were harvested and de-proteinized were then treated with E2 for a longer time period to measure with 5% 5-sulfosalicylic acid solution (SSA) (Sigma-Aldrich). apoptosis-related genes during the time when they appear to die. Total glutathione [reduced glutathione (GSH) plus glutathione MCF-7:2A cells increase both TNFα (33.55±12.09 fold change) disulfide (GSSG)] was measured spectroscopically at 412 nm and BCL2L11 (3.71±0.35 fold change) after 12 days of 1 nM using a Glutathione Assay Kit (CS0260, Sigma-Aldrich) and E2 treatment (Fig. 4B). The upregulated apoptosis-related genes the manufacturer's instructions. correspond to the time when cell death is most apparent in both cell lines, during week one in MCF-7:5C cells, and during week ROS assay.